Novel adamts-13 mutant

ABSTRACT

An enhanced disintegrin-like domain, and metalloprotease, with an isolated human thrombospondin type 1 motif, member 13 (ADAMTS-13) that includes substitutions at one or more positions in the isolated human ADAMTS-13.

This application is a divisional application of U.S. application Ser.No. 12/666,051, allowed, and incorporated herein by reference, which wasa National Stage of PCT/JP08/061211 filed Jun. 19, 2008 and claims thebenefit of JP 2007-164531 filed Jun. 22, 2007, JP 2008-020012 filed Jan.31, 2008, and JP 2008-020177 filed Jan. 31, 2008.

TECHNICAL FIELD

The present invention relates to a mutant of ADAMTS-13 which is anenzyme capable of cleaving von Willebrand factor (hereinafter referredto as “VWF”) at Tyr-Met site in A2 domain. Specifically, the presentinvention relates to a method of preparing ADAMTS-13 mutant having anenhanced enzymatic activity or a reduced reactivity to a neutralizingautoantibody by substituting amino acid residues specific to ADAMTS-13.In addition, the present invention relates to a mutant obtained by saidmethod, a pharmaceutical composition comprising as an active ingredientsaid mutant, and a pharmaceutical preparation for treating thrombosiscomprising said pharmaceutical composition.

BACKGROUND ART

ADAMTS-13 is a zinc-metalloprotease belonging to ADAMTS (adisintegrin-like domain, and metalloprotease, with thrombospondin type 1motif) family and specifically cleaves von Willebrand factor (VWF) atTyr1605-Met1606, which corresponds to 842-843 after cleavage of apreprosequence (VWF cleavage enzyme: VWF-Cleaving Protease, VWF-CP)(seee.g. Non-patent reference 1). ADAMTS-13 is known to be an activitymodulation factor of VWF that is an important factor of plateletaggregation. VWF released through stimulation or circulating in blood isimportant in forming platelet thrombus because it plays a role as acollaboration with collagen on platelet adhesion and agglutination inthe subendothelial tissue of a damaged vascular wall (e.g. seeNon-patent reference 2).

It is considered that VWF is subjected to conformational change byintravascular shear force in blood circulation to thereby expose A2domain, and Tyr1605-Met1606 therein, ADAMTS-13 cleavage site, is rapidlyhydrolyzed by ADAMTS-13. Anderson et al. evaluated a catalyticefficiency of ADAMTS-13 using as a substrate VWF previously subjected toguanidinium hydrochloride to induce the conformational change (see e.g.Non-patent reference 3). The result showed that kcat was up to 0.83min⁻¹ and kcat/Km was 55 μM⁻¹ min⁻¹, which are lower than those of theother enzymes of a coagulation system to natural high molecular weightsubstrates, indicating that ADAMTS-13 has a lower enzymatic activity.

Thrombotic thrombocytopenic purpura (hereinafter also referred to as“TTP”) caused by a reduced activity of ADAMTS-13 is classified into acongenital TTP and an acquired TTP. The congenital TTP is hereditary andcaused by a molecular abnormality of ADAMTS-13 (also called UpshowSchulmann syndrome (USS)) and the acquired TTP is positive in aneutralizing autoantibody to ADAMTS-13. While a plasma transfusion iscurrently conducted to supplement ADAMTS-13 for treating the congenitalTTP, it is desired that an ADAMTS-13 concentrate or a recombinantformulation is alternatively used for said treating in future. Moreover,a plasmapheresis is generally conducted to both remove the neutralizingantibody and to supplement ADAMTS-13 for treating the acquired TTP.

It has often reported that the molecular abnormality of ADAMTS-13 isfound in patients with congenital TTP (USS) wherein a missense and/ornonsense mutation is found throughout the molecule. However, thepathogenesis of TTP starting in adult are also found, suggesting thatpossibly the congenital reduced ADAMTS-13 is not the only trigger of thepathogenesis of TTP. Considering that there are cases of thepathogenesis of congenital TTP during a pregnancy (VWF in blood mayincrease to 300% of the normal value during late pregnancy), thesystemic platelet thrombus formation arising from TTP may be caused bythe increased VWF in blood induced by the second trigger such as anenvironment factor or a genetic factor in addition to the reducedADAMTS-13 in blood. Indeed, the present inventors have found that are-event rate of acute myocardial infarction (hereinafter also referredto as “AMI”) within 1 year is significantly high when the ratio betweenVWF and ADAMTS-13 in blood (VWF/ADAMTS-13) after 24 hours of the onsetof AMI exceeds a certain value (see e.g. Patent reference 1).

In addition, an ADAMTS-13 antigen level in plasma of patients withthrombotic disease, including disseminated intravascular coagulation(DIC), hemolytic-uremic syndrome (HUS), deep vein thrombosis (DVT), TTP,pulmonary embolism, cerebral infarction and systemic lupus erythematosus(SLE), is significantly reduced compared to healthy adult (see e.g.Patent reference 2), wherein an ADAMTS-13 antigen level is measured withsandwich ELISA using a monoclonal antibody. Moreover, with respect toDIC associated with septicemia, it was shown that patients havingADAMTS-13 blood level of less than 20% is significantly likely todevelop nephropathy as compared to patients having ADAMTS-13 blood levelof 20% or more (see e.g. Non-patent reference 4).

Meanwhile, endothelial cells stimulated by inflammatory cytokine, IL-8and/or TNF-α, induce a release of unusually large (UL) VWF that has alarger multimer structure than normal. There is also an experimentalresult that IL-6 inhibits a VWF cleaving activity of ADSAMTS-13 under ashear stress of blood Flow (ex vivo). Therefore, it is suggested thatthere is an association between inflammation and thrombus formation (seee.g. Non-patent reference 5).

In addition, it is reported that ciga toxin which causes HUS stimulatesvascular endothelial cells to promote ULVWF release and inhibits theactivity of ADAMTS-13. It seems possible that HUS is improved byadministering ADAMTS-13 to patients with HUS (see e.g. Non-patentreference 6). Thus, when the disease as stated above may be aggravateddue to an imbalance involving the reduced ADAMTS-13 and the elevatedVWF, administration of ADAMTS-13 may alleviate the condition of thedisease.

ADAMTS-13 as previously described belongs to a metalloprotease groupcalled ADAMTS family. ADAMTS-1 to -20 are known as a member of thisfamily (ADAMTS-5 is identical to ADAMTS-11). ADAMTS-13 as well as theother members of ADAMTS family has a multidomain structure (FIG. 1). Theamino acid sequence of ADAMTS-13 is encoded by a DNA which contains 4284bases and ranges from a start codon ATG(Met) to a stop codon TGA. AnADAMTS-13 gene has 29 Exons on the chromosome 9q34 and 37 kb infull-length. It is revealed via the gene sequencing of ADAMTS-13 proteinthat ADAMTS-13 protein has 1427 amino acid residues in the precursorthereof and 10 asparagine-linked glycosylation potential sites and is alarge single-strand glycoprotein (Patent reference 4).

In a process of biosynthesis, a preprosequence of 74 residues is cleavedby a processing endoprotease Furin to provide a mature form containing1353 amino acid residues. RQRR sequence (SEQ ID NO: 80), a cleaved motifof Furin, at the end of preprosequence is followed by a metalloproteasedomain that contains a Reprolysin type zinc chelate domain comprising aconsensus sequence HEXXHXXGXXHD (SEQ ID NO: 81). Then, via adisintegrin-like domain which is found in a snake venom metalloprotease,there follows the first Tsp1 motif (Tsp1-1) consisting of about 50 to 60residues which is generally thought to be important for a molecularrecognition and then a cysteine-rich domain containing one of a celladherence motif, Arg-Gly-Asp (RGD) sequence. A spacer domain containingabout 130 amino acid residues without cysteine residue then follows, andagain Tsp1 motif is repeated (Tsp1-2 to Tsp1-8) followed by CUB 1 and 2domains that have firstly been found in complement components C1r orC1s. These CUB domains are characteristic of ADAMTS-13 because amongADAMTS family only ADAMTS-13 has these domains. The present inventorshave previously identified from the metalloprotease domain to spacerdomain of ADAMTS-13 as domains essential for exerting an enzymaticactivity or as an epitope for antibody neutralization (see e.g. Patentreference 3, and Non-patent references 7 and 8).

-   Patent reference 1: JP-A-2007-248395-   Patent reference 2: WO2005/062054-   Patent reference 3: WO2004/029242-   Patent reference 4: WO2002/088366-   Non-patent reference 1: Soejima, K., Mimura, N., Hirashima, M.,    Maeda, H., Hamamoto, T., Nakagaki, T. & Nozaki, C.: A novel human    metalloprotease synthesized in the liver and secreted into the    blood: possibly, the von Willebrand factor-cleaving protease? J.    Biochem., 130: p. 475-480, 2001-   Non-patent reference 2: Soejima, K. & Nakagaki, T.: Interplay    between ADAMTS13 and von Willebrand factor in inherited and acquired    thrombotic microangiopathies. Semin. Hematol., 42: p. 56-62, 2005-   Non-patent reference 3: Anderson, P. J., Kokame, K. & Sadler, J. E.:    Zinc and calcium ions cooperatively modulate ADAMTS13 activity. J.    Biol. Chem., 281: p. 850-857, 2006-   Non-patent reference 4: Ono, T., Mimuro, J., Madoiwa, S., Soejima,    K., Kashiwakura, Y., Ishiwata, A., Takano, K., Ohmori, T. & Sakata,    Y.: Severe secondary deficiency of von Willebrand factor-cleaving    protease (ADAMTS13) in patients with sepsis-induced disseminated    intravascular coagulation: its correlation with development of renal    failure. Blood, 107: p. 528-534, 2006-   Non-patent reference 5: Bernardo, A., Ball, C., Nolasco, L.,    Moake, J. F. & Dong, J. F.: Effects of inflammatory cytokines on the    release and cleavage of the endothelial cell-derived ultralarge von    Willebrand factor multimers under flow. Blood, 104: p. 100-106, 2004-   Non-patent reference 6: Nolasco, L. H., Turner, N. A., Bernardo, A.,    Tao, Z., Cleary, T. G., Dong, J. F. & Moake, J. L.: Hemolytic uremic    syndrome-associated Shiga toxins promote endothelial-cell secretion    and impair ADAMTS13 cleavage of unusually large von Willebrand    factor multimers. Blood, 106: p. 4199-4209, 2005-   Non-patent reference 7: Soejima, K., Matsumoto, M., Kokame, K.,    Yagi, H., Ishizashi, H., Maeda, H., Nozaki, C., Miyata, T.,    Fujimura, Y. & Nakagaki, T.: ADAMTS-13 cysteine-rich/spacer domains    are functionally essential for von Willebrand factor cleavage.    Blood, 102: p. 3232-3237, 2003-   Non-patent reference 8: Soejima, K., Nakamura, H., Hirashima, M.,    Morikawa, W., Nozaki, C. & Nakagaki, T.: Analysis on the molecular    species and concentration of circulating ADAMTS13 in blood. J.    Biochem., 139: p. 147-154, 2006

DISCLOSURE OF THE INVENTION Technical Problem to be Solved by theInvention

Thus, the present invention enables the broad application as describedabove by preparing ADAMTS-13 mutant having a higher activity. Moreover,the mutant which has a reduced reactivity to a neutralizing antibodywhile holding the enzymatic activity may be applied to the acquired TTP.ADAMTS-13 mutant having such properties has never been reported so far.

Therefore, the technical problem to be solved by the present inventionis to provide ADAMTS-13 mutant having an efficiently high activity fortreating TTP etc. or a reduced reactivity to a neutralizing antibody.

Means for Solving the Problems

Under these circumstances, the present inventors have earnestly studiedto develop ADAMTS-13 mutant having a high enzymatic activity or themutant maintaining the enzymatic activity while the reactivity thereofwith a neutralizing antibody is reduced by altering an epitope for aneutralizing antibody to thereby complete the present invention. Thepresent inventors have succeeded in preparing ADAMTS-13W688X proteinmutant which has an enhanced enzymatic activity or a reduced reactivitywith a neutralizing antibody (hereinafter also referred to as “ADAMTS-13mutant”) by substitution of an amino acid contained in thedisintegrin-like domain, the cysteine-rich domain or the spacer domainof a C-terminal deletion mutant W688X. Said C-terminal deletion mutantW688X (hereinafter also referred to as “ADAMTS-13W688X protein”; seePatent reference 3 and Non-patent reference 7) is a minimum unit ofADAMTS-13 exerting the activity and is provided by deleting amino acidresidues ranging from the 689th amino acid to the C terminus from the1427 amino acid residues. That is, the present invention is as describedbelow. In addition, the modification according to the present inventionmay also be accomplished using a full-length of ADAMTS-13 (wild typestrain) molecule.

(1) ADAMTS-13 mutant wherein at least one charged amino acid in adisintegrin-like domain, a cysteine-rich domain or a spacer domain ofADAMTS-13 other than the following amino acids is substituted with adifferent amino acid: arginine at position 326, aspartic acid atposition 330, aspartic acid at position 343 and arginine at position 349in the disintegrin-like domain, aspartic acid at position 480, arginineat position 488, arginine at position 498, arginine at position 507,aspartic acid at position 533 and aspartic acid at position 543 in thecysteine-rich domain, and glutamic acid at position 641 and arginine atposition 660 in the spacer domain.

(2) The mutant according to (1) wherein said different amino acid is anuncharged amino acid.

(3) The mutant according to (1) or (2) wherein the charged amino acid tobe substituted is an amino acid present in or around the neutralizingantibody recognition epitope including at least one of arginine atposition 312, lysine at position 318, arginine at position 568, glutamicacid at position 569, arginine at position 589, lysine at position 608,glutamic acid at position 634 or aspartic acid at position 635.

(4) The mutant according to (1) or (2) wherein the charged amino acid tobe substituted is selected from the group consisting of aspartic acid atposition 298, arginine at position 312, arginine at position 326,glutamic acid at position 327, arginine at position 370, arginine atposition 452, aspartic acid at position 504, arginine at position 514,aspartic acid at position 537, arginine at position 568 and arginine atposition 659.

(5) The mutant according to any one of (2)-(4) wherein the unchargedamino acid is selected from the group consisting of alanine, glycine,proline, serine and threonine.

(6) A method of enhancing an enzymatic activity of ADAMTS-13 wherein atleast one charged amino acid in a disintegrin-like domain, acysteine-rich domain or a spacer domain of ADAMTS-13 other than thefollowing amino acids is substituted with a different amino acid:arginine at position 326, aspartic acid at position 330, aspartic acidat position 343 and arginine at position 349 in the disintegrin-likedomain, aspartic acid at position 480, arginine at position 488,arginine at position 498, arginine at position 507, aspartic acid atposition 533 and aspartic acid at position 543 in the cysteine-richdomain, and glutamic acid at position 641 and arginine at position 660in the spacer domain.

(7) A method of reducing a reactivity of ADAMTS-13 to an anti-ADAMTS-13neutralizing antibody, wherein at least one charged amino acid in adisintegrin-like domain, a cysteine-rich domain or a spacer domain ofADAMTS-13 other than the following amino acids is substituted with adifferent amino acid: arginine at position 326, aspartic acid atposition 330, aspartic acid at position 343 and arginine at position 349in the disintegrin-like domain, aspartic acid at position 480, arginineat position 488, arginine at position 498, arginine at position 507,aspartic acid at position 533 and aspartic acid at position 543 in thecysteine-rich domain, and glutamic acid at position 641 and arginine atposition 660 in the spacer domain.

(8) The method according to (6) or (7) wherein the different amino acidis an uncharged amino acid.

(9) The method according to any one of (6)-(8) wherein the charged aminoacid to be substituted is present in or around the neutralizing antibodyrecognition epitope, including at least one of arginine at position 312,lysine at position 318, arginine at position 568, glutamic acid atposition 569, arginine at position 589, lysine at position 608, glutamicacid at position 634, aspartic acid at position 635 or arginine atposition 639.

(10) The method according to any one of (6)-(8) wherein the chargedamino acid to be substituted is selected from the group consisting ofaspartic acid at position 298, arginine at position 312, arginine atposition 326, glutamic acid at position 327, arginine at position 370,arginine at position 452, aspartic acid at position 504, arginine atposition 514, aspartic acid at position 537, arginine at position 568and arginine at position 659.

(11) The method according to any one of (8)-(10) wherein the unchargedamino acid is selected from the group consisting of alanine, glycine,proline, serine and threonine.

(12) A pharmaceutical composition containing as an active ingredient theADAMTS-13 mutant of any one of (1)-(5).

(13) A therapeutic agent for thrombotic disease comprising as an activeingredient the ADAMTS-13 mutant of any one of (1)-(5).

(14) The therapeutic agent according to (13) wherein the thromboticdisease is disseminated intravascular coagulation (DIC),hemolytic-uremic syndrome (HUS), deep vein thrombosis (DVT), thromboticthrombocytopenic purpura (TTP), myocardial infarction, pulmonaryembolism, cerebral infarction or systemic lupus erythematosus (SLE).

Effects of the Invention

The present invention provides a method of preparing ADAMTS-13 mutanthaving a high enzymatic activity and the ADAMTS-13 mutant obtained bysaid method. For example, ADAMTS-13 mutant is provided by substitutingat least one of charged amino acids present in the disintegrin-likedomain, the cysteine-rich domain or the spacer domain with an unchargedamino acid. Moreover, ADAMTS-13 mutant having a reduced reactivity to aneutralizing antibody in addition to the high enzymatic activity isprovided by substituting a charged amino acid present in or around theepitope for an anti-ADAMTS-13 neutralizing antibody with an unchargedamino acid. Therefore, ADAMTS-13 mutant of the present invention may bea useful active ingredient for treating patients with thrombosis andexpected to provide a good effect with a small dosage. The term of“uncharged amino acid” as used herein refers to all of the amino acidsother than charged amino acids (lysine, arginine, glutamic acid andaspartic acid).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a gene structure and a protein domain structure ofADAMTS-13. Black dots indicate 10 N-linked glycosylation potentialsites. FIG. 1 discloses “RQRR” as SEQ ID NO: 80.

FIG. 2 shows the results of Western blot for culture supernatant ofADAMTS-13 mutant.

FIG. 3 shows evaluation of a cleaving activity of ADAMTS-13 mutant to asynthetic substrate FRETS-VWF73. The specific activity (U=units/μg) isshown as a hydrolytic activity of the mutant (units) per the amount ofprotein of the mutant (the amount of antigen) (μg). The relativeactivity (Umutant/Uwt) was obtained from a ratio of the specificactivity of the mutant to that of the wild type strain(U=U(mutant)/U(wild type strain)). The specific activity (U=units/μg);U=the hydrolytic activity of the mutant (units)/the amount of protein ofthe mutant (the amount of antigen) (μg). The relative activity(Umutant/Uwt); U mutant/U wild type strain.

FIG. 4 shows evaluation of a cleaving activity of ADAMTS-13 mutant to anatural substrate VWF. The specific activity (U=units/μg) is shown asthe hydrolytic activity of the mutant (units) per the amount of proteinof the mutant (the amount of antigen) (μg). The relative activity(Umutant/Uwt) was obtained from a ratio of the specific activity of themutant to that of the wild type strain (U=U mutant/U wild type strain).The specific activity (U=units/μg); U=the hydrolytic activity of themutant (units)/the amount of protein of the mutant (the amount ofantigen) (μg). The relative activity (Umutant/Uwt); U mutant/U wild typestrain.

FIG. 5 shows evaluation of a binding ability of ADAMTS-13 mutant to VWFimmobilized on ELISA plate. The specific activity (U=units/μg) is shownas the binding activity of the mutant (units) per the amount of proteinof the mutant (the amount of antigen)(pg). The relative activity(Umutant/Uwt) was obtained from a ratio of the specific activity ofmutant to that of the wild type strain (U=U mutant/U wild type strain).The specific activity (U=units/μg); U=the binding activity of the mutant(units)/the amount of protein of the mutant (the amount of antigen)(μg). The relative activity (Umutant/Uwt); U mutant/U wild type strain.

FIG. 6 shows the results of Western blot for an epitope analysis of aneutralizing murine monoclonal antibody. The arrows indicate theestimated epitope amino acids.

FIG. 7 shows the results of Western blot for an epitope analysis of anautoantibody of acquired TTP patient A. The arrows indicate theestimated epitope amino acids.

FIG. 8 shows the results of Western blot for an epitope analysis of anautoantibody of acquired TTP patient B. The arrows indicate theestimated epitope amino acids.

FIG. 9 shows the results of Western blot for an epitope analysis of anautoantibody of acquired TTP patient C. The arrows indicate theestimated epitope amino acids.

FIG. 10 shows evaluation of the cleaving activity of ADAMTS-13 mutantwhich has a reduced reactivity to an acquired TTP autoantibody to thesynthetic substrate FRETS-VWF73.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is characterized by a method of enhancing anenzymatic activity of ADAMTS-13 and/or a method of reducing thereactivity to an anti-ADAMTS-13 neutralizing antibody as well as amutant of ADAMTS-13 prepared by said methods. The method of the presentinvention is accomplished by substituting a charged amino acid (arginine(R), lysine (K), glutamic acid (E), aspartic acid (D)) in thedisintegrin-like domain, the cysteine-rich domain or the spacer domainwith a different amino acid, especially an uncharged amino acid on thebasis of the minimum unit of ADAMTS-13 exerting the activity(ADAMTS-13W688X protein), and evaluating the enzymatic activity of theresulting mutant and the reactivity thereof to a neutralizing monoclonalantibody. Detail of the method is described herein below.

The gene W688X encoding the minimum unit of ADAMTS-13 exerting theactivity (hereinafter also referred to as “ADAMTS-13W688X gene”) may beobtained, for example, by designing a PCR primer on the basis of thesequence as described in Non-patent reference 7 and Patent reference 3and conducting PCR using as a template cDNA derived from human organs orcells producing ADAMTS-13. In particular, the ADAMTS-13W688X gene may beprepared as described below. First, total RNAs are extracted from humanhepatocytes and then mRNAs are purified therefrom. The resulting mRNAsare converted to cDNAs, then PCR reaction is conducted using PCR primersdesigned depending on each of the gene sequences, and the resulting PCRproducts are incorporated into a plasmid vector which is introduced intoE. coli. The clone containing cDNA encoding the desired protein ischosen among E. coli colonies. For the extraction of total RNAs,commercially available reagents such as TRIzol reagent (GIBCO BRL) andISOGEN (NIPPON GENE Co., Ltd.) may be used. For the purification ofmRNAs, commercially available kits such as mRNA Purification Kit(Amersham BioSciences) may be used. For the conversion to cDNAs,commercially available kits for preparing cDNA library such asSuperScript plasmid system for cDNA synthesis and plasmid cloning (GIBCOBRL) may be used. For practically obtaining the ADAMTS-13W688X gene, acommercially available cDNA library such as e.g. Human LiverMarathon-Ready cDNA (BC Bioscience) may be used. The PCR primers arereadily available from companies in charge of DNA synthesis (e.g.QIAGEN). It is preferred that KOZAK sequence (Kozak M, J. Mol. Biol.,196, 947 (1987)) and an adequate sequence of a restriction enzymecleavage site is added to the 5′ side of the primer. The PCR reactionmay be conducted using a commercially available Advantage HF-2 PCR Kit(BC Bioscience) in accordance with the appended protocol. The basesequence of DNA fragments obtained from PCR is determined by a DNAsequencer, e.g. CEQ2000XL DNA Analysis System (Beckman) after thecloning using a TA cloning kit (Invitrogen Corporation) etc.

In order to introduce a point mutation to the resulting ADAMTS-13W688Xgene, site-directed mutagenesis may generally be used. Practically, theintroduction of a point mutation to the ADAMTS-13W688X gene is conductedusing a commercially available kit such as Site-Directed MutagenesisSystem (Takara: Mutan-Super Express Km, Mutan-Express Km, Mutan-K andthe like), QuickChange Multi Site-Directed Mutagenesis Kit, QuickChangeXL Site-Directed Mutagenesis Kit (Stratagene) and GeneTailorSite-Directed Mutagenesis System (Invitrogen) applying said technique inaccordance with the appended protocol. A total of 78 primers as listedin Table 1 may be used in order to substitute any of the charged aminoacids, N-linked glycosylation potential sites in the disintegrin-likedomain, the cysteine-rich domain, and the spacer domain of ADAMTS-13with alanine. Multiple point mutations may be introduced by repeatingsite-directed mutagenesis using as a template the ADAMTS-13W688X genewhere a point mutation is introduced and different primers. A DNAsequence of the ADAMTS-13W688X gene where a point mutation is introducedmay be determined by the above DNA sequencer.

The ADAMTS-13W688X gene or the ADAMTS-13W688X gene with a point mutation(hereinafter also referred to as “altered ADAMTS-13W688X gene”) may beincorporated into an appropriate expression vector and a host may betransformed with said expression vector to allow for expression of theminimum unit of ADAMTS-13 exerting the activity (ADAMTS-13W688X protein)and the mutant thereof (ADAMTS-13 mutant). As a host, a bacterial cell,a yeast cell, an animal cell, a plant cell and an insect cell etc. maybe used as commonly used to express a foreign protein as long asefficacy as a therapeutic agent for thrombotic disease is exhibited.Preferably, a eukaryotic cell such as an animal cell may be used. Whenan animal cell is used as a host, an expression vector may beincorporated with a promoter and marker genes for selection and for geneamplification depending on the host animal cell as used. For example,for an expression vector constructed using an expression plasmidcontaining chicken β-actin promoter (pCAGG vector), HeLa cell, 293 cell,CHO cell, or BHK cell may be used.

The known method may be used to transform a host cell. For example, theuse of calcium phosphate, the use of DEAE dextran, the method usingliposomes of Lipofectin, protoplast polyethylene glycol fusion,electroporation, and the like may be employed and appropriately beselected depending on a host cell as used (Molecular Cloning (3rd Ed.),Vol. 3, Cold Spring Harbor Laboratory Press (2001)).

Selection and proliferation of the transformed cells may be performed bya method as usually used for transformation of animal cells. Forexample, the cells after transformation may be cultured using aselection medium at 37° C. for 10-14 days while appropriately exchangingthe culture medium. The selection medium includes a medium generallyused to cultivate animal cells such as a serum-free medium, e.g.CHO-S-SFMII medium (GIBCO-BRL), IS CHO-V medium (IS Japan Co., Ltd.),YMM medium or a serum medium, e.g. MEM α medium, RPMI medium or DulbeccoMEM medium (all from GIBCO-BRL) supplemented with about 5-10% fetalbovine serum. The selection medium may contain methotrexate, G418, andthe like depending on the employed selection marker. Upon the culture,untransformed cells are not alive and only transformed cells are grown.In addition, the transformed cells are subjected to limiting dilutionetc. to select and clone cell strains of interest producing theADAMTS-13W688X protein or the ADAMTS-13 mutant.

To purify the ADAMTS-13W688X protein or the ADAMTS-13 mutant from thecells producing said protein, a purification method generally used inthe protein chemistry may be used. The purification method includes, forexample, centrifugation, salting-out, ultrafiltration, isoelectricprecipitation, electrophoresis, ion-exchange chromatography, gelfiltration, affinity chromatography, hydrophobic chromatography,hydroxyapatite chromatography and CS resin chromatography in combinationthereof. An amount of the obtained protein may be determined using areagent for protein measurement such as BCA Protein Assay Reagent Kit(Pierce Biotechnology, Inc), Protein Assay Kit (BIO-RAD, Inc), and thelike.

The ADAMTS-13W688X protein or the ADAMTS-13 mutant may be expressed inthe form of a fusion with other polypeptide or peptide to allow foreasier purification. A vector expressing such a fusion protein includesthe system which may express a fusion protein associated with FLAG tag(SIGMA), a GST fusion protein purification system which can prepare afusion protein with glutathione S transferase (GST) (AmershamPharmacia), an HAT protein expression/purification system (ClontechInc.) which is capable of adding oligohistidine, Magne His ProteinPurification System (Promega Inc), and the like. For example, asdescribed in Examples of the present invention, the ADAMTS-13 mutantexpression product expressed as a fusion protein with FLAG tag isspecifically purified using agarose gel immobilized with an anti-FLAGM2monoclonal antibody (SIGMA CORPORATION). The detection of theADAMTS-13W688X protein or the ADAMTS-13 mutant may be conducted by amethod on the basis of a molecular size such as SDS-PAGE, gelfiltration, and the like or a method on the basis of an antigen-antibodyreaction such as ELISA, Western blot, dot blot, and the like. The abovemethods are all commonly used to determine a foreign protein and may beselected in accordance with the purpose.

To evaluate the ability of the ADAMTS-13 mutant as an enzyme, theactivity to bind to or degrade von Willebrand factor (VWF) derived fromhuman plasma or partially synthesized peptide of VWF may be measured bya method such as ELISA and the like using an antibody to ADAMTS-13 or anantibody to FLAG and compared to the activity of a wild-typeADAMTS-13W688X protein with no amino acid substitution. ELISA may beconstructed by a common procedure. VWF derived from human plasma and anantibody to ADAMTS-13 for ELISA may be obtained according to the methodsof Non-patent reference 1 and Non-patent reference 8, respectively. Acommercially available fluorescently-labeled FRETS-VWF73 (PEPTIDEINSTITUTE, INC.) may be used as a partially synthesized peptide of VWF.The resulting ADAMTS-13 mutant having a higher enzymatic activity thanthe ADAMTS-13W688X protein is substituted with charged amino acids otherthan the following amino acids with alanine: aspartic acid at position343, arginine at position 349, aspartic acid at position 480, arginineat position 488, arginine at position 498, arginine at position 507,aspartic acid at position 533, aspartic acid at position 543, glutamicacid at position 641 and arginine at position 660. The number ofsubstitution of the charged amino acids may be at least one. TheseADAMTS-13 mutants may be greatly efficacious at a low dosage in human asa therapeutic agent for e.g. thrombosis. In addition, due to the lowdosage, reduction in adverse side effects may be expected.

These effects become immense by the reduced reactivity to a neutralizingautoantibody found in patients with thrombosis in addition to theelevated enzymatic activity. These ADAMTS-13 mutants may be obtained bystudying an extent of binding of the ADAMTS-13 mutant having a highenzymatic activity to a neutralizing antibody using ELISA or WesternBlotting (hereinafter also referred to as “WB”). The neutralizingantibody as used herein may be a polyclonal antibody or a monoclonalantibody. To obtain the monoclonal antibody, antibody producing cellssuch as splenocytes or lymphocytes may be collected from an animalimmunized with mature dendritic cells from immature dendritic cells, theresulting cells may be fused with myeloma cell line according toMilstein et al., Method Enzymol., 73, 3-46, 1981, to prepare hybridomaswhich produce an antibody to a specific antigen. Alternatively, anantibody preparation technique using a phage display (Phage Display ofPeptides and Proteins: A Laboratory Manual Edited by Brian K. Kay etal., Antibody Engineering: A PRACTICAL APPROACH Edited by J. McCAFFERTYet al., ANTIBODY ENGINEERING second edition edited by Carl A. K.BORREBAECK) may be used to prepare an antibody which binds to a specificantigen. For the above ELISA or WB, the resulting antibody may belabeled by fluorescence labeling, RI, biotinylation, and the like. Theseare all commercially available as a kit.

The resulting ADAMTS-13 mutant is preferably such that the charged aminoacids comprising at least one of arginine at position 312, lysine atposition 318, arginine at position 568, glutamic acid at position 569,arginine at position 589, lysine at position 608, glutamic acid atposition 634, aspartic acid at position 635 or arginine at position 639in or around the neutralizing antibody recognition epitope aresubstituted with alanine. Alternatively, the ADAMTS-13 mutant ispreferably such that the charged amino acids selected from the groupconsisting of aspartic acid at position 298, arginine at position 312,arginine at position 326, glutamic acid at position 327, arginine atposition 370, arginine at position 452, aspartic acid at position 504,arginine at position 514, aspartic acid at position 537, arginine atposition 568 and arginine at position 659 are substituted. As describedabove, the number of substitution of the charged amino acids may be 1 ormore.

Moreover, the charged amino acids may be substituted with not onlyalanine as in Examples but also with other uncharged amino acids,preferably glycine, proline, serine and threonine which are subject toeasy amino acid substitution during the course of evolution (i.e.analogous amino acids)(BIOINFORMATICS A Practical Guide to the Analysisof Genes and Proteins Edited by ANDREAS D. BAXEVANIS, B. F. FRANCISOUELLETTE, John Wiley & Sons, Inc.).

The ADAMTS-13 mutant of the present invention may be formulated into apharmaceutical preparation for treatment, diagnosis or other uses. Forexample, to prepare a preparation for intravenous administration, acomposition may generally be dissolved in an aqueous solution thatcontains physiologically compatible materials such as sodium chloride,glycine etc. and has a physiologically compatible, buffered pH. Alyophilized formulation may be employed as a final form to enableprolonged stability. A guideline for a composition for intravenousadministration is established by the Government's regulation, e.g.“Minimum Requirements for Biological Products”. A pharmaceuticalcomposition of the present invention comprising as an active ingredientthe ADAMTS-13 mutant may specifically be used in patients having areduced ADAMTS-13 level such as patients with disseminated intravascularcoagulation (DIC), hemolytic-uremic syndrome (HUS), deep vein thrombosis(DVT), thrombotic thrombocytopenic purpura (TTP), myocardial infarction,pulmonary embolism, cerebral infarction, systemic lupus erythematosus(SLE), and the like. Alternatively said pharmaceutical composition maybe used as a supplemental treatment in patients with an elevated bloodconcentration of VWF, a substrate of this enzyme, or in patientsforeseen to develop ULVWF due to inflammation etc.

The present invention is explained by the following Examples which arenot construed to limit the present invention. The present invention isspecifically exemplified by Examples together with the appendeddrawings. In Examples, the mutants were expressed in culture supernatantof animal cells (HeLa). Unless otherwise instructed, reagents anddevices etc. used for genetic recombination were available from TaKaRaSHUZO, Toyobo, Bio-Rad, PerkinElmer Applied, Beckman and New EnglandBiolabs.

EXAMPLE 1 Preparation of Alanine Substituents

A DNA comprising the coding region in pCR vector of the gene W688Xencoding the minimum unit exerting the ADAMTS-13 activity(ADAMTS-13W688X gene) as described in Patent reference 3 withintroduction of FLAG tag sequence at the C-terminal was digested withrestriction enzymes XhoI/SalI and extracted. The resulting product wasrecloned into pKF vector attached to Site-Directed Mutagenesis kitMutan-Super Express Km (TaKaRa). According to the package insert of thekit, the 5′ phosphorylated synthetic DNA primer sequences listed intables 1 to 4 were prepared. With these, a total of 78 plasmids wereprepared containing the altered ADAMTS-13W688X genes encoding theprotein wherein the desired charged amino acids were substituted withalanine. All of the altered ADAMTS-13W688X genes were confirmed by theautomatic sequencer CEQ2000XL DNA Analysis System (Beckman). Table 1shows primers for preparing mutants with substitution in thedisintegrin-like domain, Table 2 for primers for preparing mutants withsubstitution in the cysteine-rich domain, Table 3 for primers forpreparing mutants with substitution in the spacer domain, and Table 4for primers for preparing deglycosylation mutants with substitution inthe cysteine-rich and spacer domains. In the primers indicated in theTables, the numerals on the left are a serial number of the primers and“R287A”, for example, following the period indicates that the amino acidresidue arginine (R) at position 287 is substituted with alanine (A). Inaddition, the mutation site is indicated with lower case in the basesequence.

TABLE 1 SEQ ID NOS: 2-21, left count to right column,respectively, in order of appearance. Primer Base Sequence PrimerBase Sequence  1. R287A GACCCGCCGgcGCCTCAACCC 11. R349AAGCTGCAGCgcCCTCCTCGTTC  2. D298A CACCCGCCGGcTGCGCAGCC 12. D356ACCTCTCCTGGcTGGGACAGAATG  3. E309A AGCGCCAACGcGCAGTGCCGC 13. E359AGATGGGACAGcATGTGGCGTG  4. R312A GAGCAGTGCgcCGTGGCCTTC 14. E363ATGTGGCGTGGcGAAGTGGTGC  5. K318A TTCGGCCCCgcGGCTGTCGCC 15. K364AGGCGTGGAGgcGTGGTGCTCC  6. R326A ACCTTCGCCgcGGAGCACCTG 16. K368ATGGTGCTCCgcGGGTCGCTGC  7. E327A TTCGCCAGGGcGCACCTGG 17. R370ATCCAAGGGTgcCTGCCGCTCC  8. D330A GAGCACCTGGcTATGTGCCAG 18. R372AGGTCGCTGCgcCTCCCTGGTG  9. D340A TGCCACACAGcCCCGCTGGAC 19. E376ATCCCTGGTGGcGCTGACCCCC 10. D343A GACCCGCTGGcCCAAAGCAGC 20. R386AGTGCATGGGgcCTGGTCTAGC

TABLE 2 SEQ ID NOS: 22-48, left column to right column,respectively, in order of appearance. Primer Base Sequence PrimerBase Sequence 21. R452A CAGTGCGCCgcGACCGACGGC 35. D516ACCCCGGGAGGcCGGGACCCTG 22. D454A GCCAGGACCGcCGGCCAGCCG 36. R528AGGCAGCTGCgcGACATTTGGC 23. R459A CAGCCGCTGgcCTCCTCCCCTG 37. D533ATTTGGCTGTGcTGGTAGGATG 24. D480A AGCCAAGGGGcTGCTCTGTGC 38. R535ATGTGATGGTgcGATGGACTCC 25. R484A GCTCTGTGCgcACACATGTGC 39. D537AGGTAGGATGGcCTCCCAGCAG 26. R488A CACATGTGCgcGGCCATTGGC 40. D543ACAGGTATGGGcCAGGTGCCAG 27. E492A GCCATTGGCGcGAGCTTCATC 41. R544AGTATGGGACgcGTGCCAGGTG 28. K497A TTCATCATGgcGCGTGGAGAC 42. D551ATGTGGTGGGGcCAACAGCACG 29. R498A ATCATGAAGgcTGGAGACAGC 43. R558ATGCAGCCCAgcGAAGGGCTC 30. D500A AAGCGTGGAGcCAGCTTCCTC 44. K559AAGCCCACGGgcGGGCTCTTTC 31. D504A AGCTTCCTCGcTGGGACCCGG 45. R566AACAGCTGGCgcAGCGAGAG 32. R507A GATGGGACCgcGTGTATGCC 46. R568AGGCAGAGCGgcAGAATATGTC 33. R514A AGTGGCCCCgcGGAGGACGGG 47. E569AAGAGCGAGAGcATATGTCACG 34. E515A GGCCCCCGGGcGGACGGGACC

TABLE 3 SEQ ID NOS: 49-75, left column to right column,respectively, in order of appearance. Primer Base Sequence PrimerBase Sequence 48. R589A GCCAACCACgcGCCTCTCTTC 62. E642ACGCCTGGAGGcGATCCGCAC 49. R598A TTGGCGGTGgcGATCGGAGG 63. R644AGAGGAGATCgcCATCTGGGG 50. R602A ATCGGAGGGgcCTATGTCGTG 64. E651ACCCCTCCAGGcAGATGCTGAC 51. K608A GTGGCTGGGgcGATGAGCATC 65. D652ACTCCAGGAAGcTGCTGACATC 52. E622A TCCCTCCTGGcGGATGGTCG 66. D654AGAAGATGCTGcCATCCAGG 53. D623A CTCCTGGAGGcTGGTCGTGTC 67. R659ACAGGTTTACgcGCGGTATGG 54. R625A GAGGATGGTgcTGTCGAGTAC 68. R660AGTTTACAGGgcGTATGGCGAG 55. E627A GGTCGTGTCGcGTACAGAGTG 69. E663ACGGTATGGCGcGGAGTATGG 56. R629A GTCGAGTACgcAGTGGCCCTC 70. E664ATATGGCGAGGcGTATGGCAAC 57. E634A GCCCTCACCGcGGACCGGCTG 71. R670AAACCTCACCgcCCCAGACATC 58. D635A CTCACCGAGGcCCGGCTGCC 72. D672AACCCGCCCAGcCATCACCTTC 59. R636A ACCGAGGACgcGCTGCCCCG 73. K681ATTCCAGCCTgcGCCACGGCAG 60. R639A CGGCTGCCCgcCCTGGAGGAG 74. R683ACCTAAGCCAgcGCAGGCCTG 61. E641A CCCCGCCTGGcGGAGATCCG

TABLE 4 SEQ ID NOS: 76-79, respectively, in order of appearance. PrimerBase sequence 75. N552A GGTGGGGACgcCAGCACGTGC 76. N579AGTTACCCCCgcCCTGACCAG 77. N614A ATCTCCCCTGCCACCACCTAC 78. N667AGAGTATGGCGCCCTCACCCGC

EXAMPLE 2 Preparation of Vector for Mutant Expression

The expression vector pCAGG (JP Patent 2824434) was digested with SalIand ligated with the plasmid containing the altered ADAMTS-13W688X genesprepared in Example 1 previously cleaved with SalI/XhoI, followed bytransformation of E. coli JM109 therewith and incubation on the LB agarmedium containing ampicillin to select transformed E. coli cells. Theresulting colonies were incubated with a commercially available culturemedium overnight, and then extraction and purification were conducted toprepare the desired expression plasmids.

EXAMPLE 3 Expression in Culture Supernatant and Purification of Mutants

The expression vectors containing the altered ADAMTS-13W688X genes ofExample 2 were transfected into HeLa cells using a commerciallyavailable Lipofectin reagent. The culture supernatant of temporaryexpression (15 mL) was collected on Day 3 after the gene transfection.First, Western Blotting was conducted to check whether or not thesecretion was present in the culture supernatant by a common procedureusing a commercially available antibody to FLAG tag (anti-FLAGM2monoclonal antibody (SIGMA CORPORATION)). As a result, as showed in FIG.2, for four alanine substituents: R507A(32), D533A(37) and D543A(40) inthe cysteine-rich domain and E641A(61) in the spacer domain, no normalexpression in culture supernatant could be found (the numeral inparenthesis of the mutants indicates the primer number; hereinafter thesame). The culture supernatants other than these were concentrated to 1mL using Centriprep YM-10 (Millipore). Due to the FLAG tag sequencepossessed by all the mutants at their C-terminal, affinity purificationwas conducted using agarose gel with an anti-FLAGM2 monoclonal antibodyimmobilized thereon (SIGMA CORPORATION). The elution was conducted usinga FLAG peptide solution at a concentration of 100 μg/mL in accordancewith an attached protocol.

EXAMPLE 4 Measurement of Hydrolytic Activity of Mutants to VWF PartialPeptide Substrate (FRETS-VWF73)

The activity of each of the mutants of Example 3 was measured using thecommercially available fluorescent substrate FRETS-VWF73 (PEPTIDEINSTITUTE, INC.). A concentration of the purified protein was normalizedby sandwich ELISA using the anti-human ADAMTS-13 rabbit polyclonalantibody described in Non-patent reference 8 and an HRP labeled form ofthe anti-FLAGM2 murine monoclonal antibody (SIGMA CORPORATION). Withthis, the fluorescent substrate cleaving activity was measured toquantify the specific activity. Finally, the mutants were evaluated fora relative activity as compared to the specific activity of the wildtype W688X with no alanine substitution, the specific activity of whichis 1. Thus, it may be concluded that when the relative activity issignificantly higher than 1, the mutant will have a higher enzymaticactivity than the wild type strain, and when the relative activity issignificantly lower than 1, the mutant will have a lower enzymaticactivity (or said amino acid is important for exerting the activity).

As a result, as showed in FIG. 3, the amino acid substitutions atposition R349 in the disintegrin-like domain and at position R498(29) inthe cysteine-rich domain led to the significantly reduced enzymaticactivity (the bar graph in FIG. 3 is accompanied by the estimatedexperimental error of ±20% of this experimental system). On the otherhand, the elevated enzymatic activity was observed for the amino acidsubstitutions at position R326(6), at position R370(17), at positionR568(46) and at position R659(67).

EXAMPLE 5 Measurement of VWF Degrading Activity of Mutants

The activity of each of the mutants of Example 3 was measured asdescribed below using as a substrate the VWF derived from human plasmapurified as described in Non-patent reference 1. Referring to Non-patentreference 3, VWF was denatured by incubation with 1.2 M guanidinehydrochloride at 37° C. for 1 h. Then, the VWF was reacted with anappropriate concentration of the ADAMTS-13 mutant at 37° C. for 1 h andthe reaction was quenched with EDTA of a final concentration of 5 mM.Thereafter, a degree of cleavage of VWF was measured by collagen bindingELISA (Gerritsen, H E., Turecek, P L., Schwarz, H P., Lammle, B. &Furlan, M.: Assay of von Willebrand factor (vWF)-cleaving protease basedon decreased collagen binding affinity of degraded vWF: a tool for thediagnosis of thrombotic thrombocytopenic purpura (TTP). Thromb. Haemost.82: 1386-1389, 1999). A concentration of the purified protein wasnormalized by sandwich ELISA using an anti-human ADAMTS-13 rabbitpolyclonal antibody described in Non-patent reference 8 and an HRPlabeled form of an anti-FLAGM2 murine monoclonal antibody (SIGMACORPORATION). With these, the fluorescent substrate cleaving activitywas measured to quantify the specific activity. Finally, the mutantswere evaluated for a relative activity as compared to the specificactivity of wild type W688X with no alanine substitution, the specificactivity of which is 1. Thus, it may be concluded that when the relativeactivity is significantly higher than 1, the mutant will have a higherenzymatic activity than the wild type strain, and when the relativeactivity is significantly lower than 1, the mutant will have a lowerenzymatic activity (or said amino acid is important to exerting theactivity).

As a result, as showed in FIG. 4, the substitutions at position R326(6),at position D330(8), at position D343(10) and at position R349(11) inthe disintegrin-like domain, at position D480(24), at position R488(26)and at position R498(29) in the cysteine-rich domain, and in thevicinity of position R660(68) in the spacer domain led to the reducedbinding ability to VWF. On the other hand, the substitutions at positionD298(2) and at position E327(7) in the disintegrin-like domain, atposition R452(21), at position D504(31), at position R514(33) and atposition D537(39) in the cysteine-rich domain led to the enhanced VWFcleaving activity (the bar graph in FIG. 4 is accompanied by theestimated experimental error of ±20% of this experimental system).

EXAMPLE 6

Evaluation of Mutants for their Binding Ability to VWF

Each of the purified mutants of Example 3 was evaluated for theirbinding ability to VWF derived from human plasma immobilized on ELISAplate as described in Non-patent reference 8. The concentration wasnormalized by sandwich ELISA using the anti-human ADAMTS-13 rabbitpolyclonal antibody and an HRP labeled form of the anti-FLAGM2 murinemonoclonal antibody (SIGMA CORPORATION) as described above and the VWFbinding ability per unit concentration was quantified. Finally, themutants were evaluated for a relative activity as compared to thespecific activity of wild type W688X with no alanine substitution, thespecific activity of which is 1. Thus, it may be concluded that when therelative activity is significantly higher than 1, the mutant will have ahigher affinity than the wild type strain, and when the relativeactivity is significantly lower than 1, the mutant will have a loweraffinity (or said amino acid is important for the recognition of VWF).

As a result, as showed in FIG. 5, the substitutions at position D343(10)and at position R349(11) in the disintegrin-like domain, at positionD480(24), at position R488(26) and at position R498(29) in thecysteine-rich domain and at position R660(68) in the spacer domain ledto the reduced binding ability to VWF. On the other hand, thesubstitutions at position E327(7) in the disintegrin-like domain led tothe enhanced affinity to VWF (the bar graph in FIG. 5 is accompanied bythe estimated experimental error of ±20% of this experimental system).

EXAMPLE 7 Epitope Analysis of Neutralizing Antibody 1

Following the Western Blotting (WB) in Example 3 to confirm theexpression, the anti-FLAG antibody was released by a WB separatingsolution (NAKARAI), reacted again with the murine monoclonal antibodies(WH2-22-1A, W688X6-1 and W688X3-69) having an ability to neutralize theADAMTS-13 activity established by the present inventor using commonprocedures as described in Non-patent reference 8, and visualized withan HRP labeled form of the anti-murine immunoglobulin (VECTOR) whereinthe epitopes of the antibodies were confirmed by a reduced colorintensity (FIG. 6). As a result, it was revealed that the vicinity ofR312 and K318 for WH2-22-1A, the vicinity of R568 and E569 for W688X6-1and the vicinity of R589 and D635 for W688X3-69 were recognized as anepitope. In addition, it was revealed that these amino acid residuesrecognized as an epitope were different from the amino acid residuesthought to be important for the activity to hydrolyze FRETS-VWF73 andfor the binding to VWF. It was suggested that the altered moleculehaving a reduced reactivity to antibodies from patients may be designedby this method without affecting the enzymatic activity through theepitope analysis of neutralizing antibodies from patients.

EXAMPLE 8 Epitope Analysis of Neutralizing Antibody 2

Following the WB (under non-reducing condition) to confirm theexpression in Example 3, the anti-FLAG antibody was released with a WBreleasing solution (NAKARAI), in a similar way to Example 7, reactedagain with immunoglobulin G (IgG) fractions isolated from acquired TTPpatients having the ability to neutralize the ADAMTS-13 activity, andvisualized with an HRP labeled form of the anti-human immunoglobulin(Dako) wherein the epitopes of the antibodies were confirmed by areduced color intensity (FIGS. 7 to 9). As a result, it was suggestedthat the vicinity of K608, E634, D635, E642 and R644 in the spacerdomain and N578, N614 and N667 in the glycosylation site were recognizedas an epitope. In addition, it was revealed that these amino acidresidues recognized as an epitope were different from the amino acidresidues thought to be important for the activity to hydrolyzeFRETS-VWF73 and for the binding to VWF. Therefore, the substitution ofthese major epitope amino acids with different amino acids allows forpreparing altered molecules having the reduced reactivity to antibodiesfrom patients while the enzymatic activity is maintained or evenenhanced like the mutant of E327.

EXAMPLE 9

Evaluation of Mutants with Reduced Reactivities with Antibodies fromPatients

The reactivity of the mutants, which were estimated from the result ofExample 8 to have the reduced reactivity with autoantibodies fromacquired TTP patients (K608A, E634A, D635A, E642A, R644A, andglycosylation site N578A, N614A, N667A), with the autoantibodies wasevaluated using the commercially available fluorescent substrateFRETS-VWF73(PEPTIDE INSTITUTE, INC.) (FIG. 10). The cleaving activity ofthe ADAMTS-13 mutants to the FRETS-VWF73 fluorescent substrate per 1min. (AFU/min) was measured with and without addition of antibodies frompatients A, B and C (Ab+/−) to select mutants having a sufficientenzymatic activity even when a neutralizing antibody from the patientsis added (i.e. the enzymatic activity kept at 500 or more of that ofwithout addition of the neutralizing antibodies). As a result, it wasrevealed that depending on patient antibody, K608A, E634A and D635A hada sufficient enzymatic activity. In addition, K608A was revealed to havea sufficient enzymatic activity to all the patient antibodies.

Therefore, the substitution of these major epitope amino acids withdifferent amino acids which reduce the antibody reactivity allows forpreparing altered molecules (e.g. double mutant such as E327A/K608A)having the reduced reactivity to antibodies from patients while theenzymatic activity is maintained or even enhanced like the E327 mutant.

INDUSTRIAL APPLICABILITY

The ADAMTS-13 mutant of the present invention may be used as amedicament significantly efficacious as a supplemental treatment tothrombotic disease such as TTP.

1. (canceled)
 2. An ADAMTS-13 (a disintegrin-like domain, andmetalloprotease, with an isolated human thrombospondin type 1 motif,member 13) mutant, comprising a substitution of the following positionsin the isolated human ADAMTS-13 with a different amino acid: asparticacid at position 287, aspartic acid at position 298, glutamic acid atposition 309, arginine at position 312, lysine at position 318, glutamicacid at position 327, aspartic acid at position 340, glutamic acid atposition 363, lysine at position 364, lysine at position 368, arginineat position 370, arginine at position 372, arginine at position 386,arginine at position 452, arginine at position 459, glutamic acid atposition 492, lysine at position 497, aspartic acid at position 500,aspartic acid at position 504, arginine at position 514, glutamic acidat position 515, aspartic acid at position 516, arginine at position535, aspartic acid at position 537, arginine at position 544, asparticacid at position 551, asparagine at position 552, arginine at position558, lysine at position 559, arginine at position 566, arginine atposition 568, glutamic acid at position 569, asparagine at position 579,lysine at position 608, asparagine at position 614, arginine at position625, arginine at position 629, glutamic acid at position 634, asparticacid at position 635, arginine at position 636, arginine at position639, arginine at position 644, glutamic acid at position 651, arginineat position 659, glutamic acid at position 663, glutamic acid atposition 664, asparagine at position 667, arginine at position 670, orarginine at position 683 in the amino acid sequence encoded by thenucleotide sequence of SEQ ID NO:1.
 3. The ADAMTS-13 mutant according toclaim 2, comprising a substitution of arginine at position 312, lysineat position 318, arginine at position 568, glutamic acid at position569, asparagine at position 579, lysine at position 608, asparagine atposition 614, glutamic acid at position 634, aspartic acid at position635 or arginine at position 639, glutamic acid at position 644, orasparagine at position
 667. 4. The ADAMTS-13 mutant according to claim2, comprising a substitution of arginine at position 312, lysine atposition 318, arginine at position 568, glutamic acid at position 569,asparagine at position 579, lysine at position 608, asparagine atposition 614, glutamic acid at position 634, aspartic acid at position635, arginine at position 639, arginine at position 644, glutamic acidat position 651, arginine at position 659, glutamic acid at position663, glutamic acid at position 664, asparagine at position 667, orarginine at position
 670. 5. The ADAMTS-13 mutant according to claim 2,wherein the different amino acid is an uncharged amino acid.
 6. TheADAMTS-13 mutant according to claim 3, wherein the different amino acidis an uncharged amino acid.
 7. The ADAMTS-13 mutant according to claim4, wherein the different amino acid is an uncharged amino acid.
 8. TheADAMTS-13 mutant according to claim 5, wherein the uncharged amino acidis selected from the group consisting of alanine, glycine, proline,serine and threonine.
 9. The ADAMTS-13 mutant according to claim 6,wherein the uncharged amino acid is selected from the group consistingof alanine, glycine, proline, serine and threonine.
 10. The ADAMTS-13mutant according to claim 7, wherein the uncharged amino acid isselected from the group consisting of alanine, glycine, proline, serineand threonine.